CN104007079A - Spectral detection method for recombinant human erythropoietin PEGylation reaction liquid and application thereof - Google Patents
Spectral detection method for recombinant human erythropoietin PEGylation reaction liquid and application thereof Download PDFInfo
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Abstract
The invention relates to the production technology field of PEGylated (polyethylene glycol) recombinant human erythropoietin (PEG-EPO), especially to a spectral detection method for a PEG-EPO reaction liquid and a rapid identification method for a plurality of reaction liquids before merging. The detection system comprises a phosphate buffer solution and a nanogold sol color developing agent, and a diluted PEG-EPO reaction liquid. The absorbance at a 260nm site is measured, and then the absorbance and a dilution factor are subjected to linear fitting to calculate the absorbance decline coefficient (k260). By comparing the k260 value of each reaction liquid, whether the PEGylation reaction rates of the reaction liquids are identical or close can be judged rapidly. And then, by combining the detection result of high performance liquid chromatography, the PEGylation reaction rates of the reaction liquids with the same k260 value can be obtained rapidly. Thus, the method provided by the invention greatly shortens the detection analysis time of the PEG-EPO reaction liquid before merging, improves the efficiency of the PEG-EPO drug production process, and saves the human cost and time cost. And the detection method provided by the invention has good sensitivity, reliability and reproducibility, and can be applied to the actual production process of PEG-EPO drugs.
Description
Technical field
The present invention relates to the production technical field of Pegylation Recombinant Human Erythropoietin, relate in particular to a kind of spectrum detection method and application thereof of Recombinant Human Erythropoietin pegylation reaction liquid.
Background technology
It is to the highly effective medicine of renal anemia disease that promoting erythrocyte generates stimulant (Erythropoiesis-stimulating Agents, ESAs).ESAs is anemia symptom fundamentally, recovers significantly patient's physical efficiency, improves patient's quality of life.In addition, elimination Anemia can reduce the M & M of other relevant disease significantly.But, because the Half-life in vivo of ESAs is relatively short, therefore patient needs on every Mondays to three time administration conventionally.In order to overcome this shortcoming, in recent years, researchist has carried out chemical modification research to the protein structure of Recombinant Human Erythropoietin (recombinant human Erythropoietin, EPO), to improve its pharmacokinetics and pharmacodynamics, thus the minimizing of permission administration frequency.Meanwhile, can with water soluble pegylation compound (the Polyethylene Glycol of protein molecule generation covalent bonding, PEG), be successfully applied to and improve the pharmacokinetics of some pharmaceutical grade proteins and reduce its immunogenicity.The EPO medicine (PEG-EPO) of modifying through PEGization, is called as long-acting EPO of new generation, also by successfully preparation (its chemical equation as shown in Figure 1), and becomes prescription drug in areas such as Europe.Relevant zooscopy data and clinical research result show, PEG-EPO has the Half-life in vivo of prolongation, compared with conventional EPO, can allow lower administration frequency, can be to be administered once fortnight, or even four weeks was administered once.This administering mode not too frequently, has given patient and health care provider a very large facility.Especially, PEG-EPO medicine can, in whole treatment cycle, maintain the stability of patient blood hemoglobin level, has reduced the generation for the treatment of spinoff.
Select suitable PEG reagent and PEGization reaction conditions, though PEGization can not produce significantly impact to the avtive spot of biomolecule or biological function, but it at least can have a certain impact conventionally, especially in the time forming polysubstituted PEGization product, probably the avtive spot of protein is produced to steric effect, reduce its external activity and even activity in vivo.And reduce the polysubstituted rate that PEGization is reacted, and improve its monosubstituted rate, and purifying obtains monosubstituted product from gained reactant liquor simultaneously, always be the Focal point and difficult point in PEGization pharmaceutical grade protein research process.Chinese patent " polymer/recombinant human erythropoietin couple " (200710123683.X), discloses a kind of preparation method of PEG-EPO medicine, does not contain polysubstituted PEG-EPO product (multi-PEG-EPO) in its final product.In the embodiment of this patent, gained final product has been carried out to zoopery, it is long-lasting that the data obtained has confirmed that it has.Relevant patent " a kind of purifying of mPEG-EPO and preparation method " (201010516922.X), discloses the purifying process of a kind of mPEG-EPO product (mono-PEG-EPO).In this purifying process, need to adopt hydrophobic chromatography method, separate multi-PEG-EPO and mono-PEG-EPO.Therefore, the content of multi-PEG-EPO in gained PEG-EPO reactant liquor, and ratio between mono-PEG-EPO and multi-PEG-EPO, overall difficulty and cost to purifying process have a great impact.In keeping the higher level of mono-PEG-EPO output, reduce the output of multi-PEG-EPO, can save significantly processing time and the Material Cost of purifying process.Another patent " reclaiming the method for hematopoietin " (201210313863.5), discloses a kind of method that reclaims unreacted epo protein matter from PEG-EPO reactant liquor.In the embodiment of this patent, carry out PEGization reaction test to reclaiming the EPO obtaining, the monosubstituted rate of gained is 35.5%, polysubstituted rate is 4.4%, has illustrated that the EPO reclaiming still has stronger PEGization reactivity.In this reaction result, although the output of mono-PEG-EPO is lower, the ratio between mono-PEG-EPO and multi-PEG-EPO is very high, and follow-up purifying process is had to obvious benefit.Further result of study shows, adopt the PEGization method of patent " the continuous pegylation reaction method of Recombinant Human Erythropoietin " (201210257542.8) disclosed EPO, can effectively gained be reclaimed to the monosubstituted rate of EPO and bring up to more than 40%, overall reaction rate is greater than 60%.This has illustrated, the free EPO in the PEGization reactant liquor of EPO still has certain PEGization reactivity.
And if make full use of as much as possible the reactivity of EPO, so just need to set up a kind of method for quick of the reactant liquor of the PEGization to EPO, reaction conditions is made to suitable adjustment, and then the further PEGization of free EPO in reactant liquor.At present, the detection method of the PEGization reactant liquor to EPO is mainly chromatographic process and electrophoresis method.For example; the people such as Hao Sujuan (SCI; 2010; 31 (11); 2239～2245) in the time of the PEGization reaction of carrying out EPO; first add excessive glycocoll cessation reaction, then high-efficient gel filtration chromatography analysis is carried out in sampling, is finally calculated monosubstituted rate and the polysubstituted rate of gained reactant liquor through software by each product peak area.The people (Chinese patent 200780037291.X) such as A A Buqiao Paderewski are in the time of the PEGization reaction of carrying out EPO, gained reactant liquor is detected with polyacrylamide gel electrophoresis method, determine that final product is the roughly equal mono-PEG-EPO of ratio and multi-PEG-EPO.In addition, Apollon Papadimitriou (US Patent No. 7169754B2) is in the time of the PEGization reaction of carrying out EPO, gained reactant liquor is undertaken after purifying by techniques such as ion-exchange chromatographies, with chromatogram and electrophoresis qualification products therefrom, the monosubstituted rate that draws the PEG-EPO reactant liquor before purifying is 40%, polysubstituted rate is 38%, and free EPO content is 20%.The people such as Li Xionghui (Chinese patent, 201010516922.X), in the time of the PEGization reaction of carrying out EPO, sample at once and do HPLC detection afterwards in reaction, result shows that the monosubstituted rate of gained PEG-EPO reactant liquor is 50%, polysubstituted rate is 18%, and free EPO content is 32%.Above detection method, although can carry out qualitative detection and quantitatively detect gained PEG-EPO reactant liquor, the problem such as all have step complexity, length consuming time, can only detect again after reaction finishes.
Simultaneously, in the large-scale production of carrying out PEG-EPO medicine, during such as the PEG-EPO reactant liquor of preparation 1800mg EPO reacting dose, can adopt the synthetic schemes of " repeatedly reaction on a small quantity; remerge and obtain a batch of reactant liquor ", first carry out the pre-reaction of a 5mg EPO reacting dose, and then carry out a 200mg and react with the PEGization of four 400mg EPO reacting doses, finally the PEGization reactant liquor of gained EPO is merged to the PEGization reactant liquor of the 1800mg EPO that obtains batch.That is to say, in the medicine production process of whole PEG-EPO, will carry out continuously the PEGization reactant liquor of 6 EPO; And the PEGization reactant liquor of the EPO that these prepare is before merging, all must detect, meet production requirement with the PEGization reactant liquor that ensures the 1800mg EPO obtaining after merging, wherein one of most important index is exactly PEGization reaction rate.Conventionally adopt the PEGization reaction rate of the PEGization reactant liquor of HPLC method to each EPO to detect, approach 1h each detection time, the quantity of PEGization reactant liquor of the EPO detecting in view of needs is larger, therefore produce one and detected and analyze longer problem consuming time, and increased PEG-EPO reactant liquor depositing the risk that mass change occurs in process.So we need to develop a kind of analytical technology that can carry out to the PEGization reaction rate of the PEGization reactant liquor of EPO fast detecting.
Summary of the invention
The technical problem to be solved in the present invention is to detect by ultraviolet spectrum the detection system being made up of the PEGization reactant liquor of nano gold sol, phosphate buffer and EPO, thereby a kind of spectrum detection method of Recombinant Human Erythropoietin pegylation reaction liquid is provided, and applies the method for carrying out Rapid identification before this spectrum detection method merges multiple Recombinant Human Erythropoietin pegylation reaction liquid.
For achieving the above object, the present invention is by the following technical solutions:
A spectrum detection method for Recombinant Human Erythropoietin pegylation reaction liquid, comprises the following steps:
S1 dilution: get n part reactant liquor sample, reactant liquor sample is diluted by different extension rates, obtain the dilution of n kind variable concentrations, and n≤3.
Preferably, described water is water for injection, described n=3, and described extension rate is respectively 4 times, 8 times and 10 times.
S2 configuration detection system: each dilution is mixed in different color comparison tubes from phosphate buffer and nano gold sol, and water is settled to scale and obtains having the detection liquid of variable concentrations, more than then detecting liquid colour developing 5min; The concentration of described phosphate buffer is 10-60mM, and pH is 5.0-8.0; The concentration of described nano gold sol is 1.0 × 10
-11-6.0 × 10
-9mol/L; The volume ratio of described phosphate buffer, nano gold sol and dilute reaction solution is 2:1:1.Described water is water for injection.
Preferably, the concentration of described phosphate buffer is 40mM, and pH is 7.0; The concentration of nano gold sol is 3.0 × 10
-9mol/L; The particle diameter of nano gold sol is 15-25nm; Detect liquid colour developing 5-20min.
S3, detection: survey the absorbance A of each detection liquid at 260nm place with ultraviolet spectrometer (UVS)
260.
S4, data processing: by the absorbance A of each detection liquid
260and extension rate carries out linear fit, obtain matched curve A
260=ax+b, the extension rate that x is reactant liquor; That gets a thoroughly deserves absorbance descent coefficient K
260.Also the absolute value of a can be multiplied by 10000 rear institute values and be made as absorbance descent coefficient K
260.
A rapid identification method before the multiple Recombinant Human Erythropoietin pegylation reaction liquid of applying above spectrum detection method merge, comprises the following steps:
S1 ultraviolet detects: each reactant liquor is carried out respectively to dilution as claimed in claim 1, configuration detection system, detection and four steps of data processing successively, obtain the absorbance descent coefficient K of each reactant liquor
260.
S2 high performance liquid chromatography detects: the pegylation reaction rate that detects one of them reactant liquor with high performance liquid chromatograph; This reactant liquor is benchmark reactant liquor.
S3, comparison: by the absorbance descent coefficient K of other reactant liquor
260absorbance descent coefficient K with benchmark reactant liquor
260relatively, there is identical absorbance descent coefficient k
260reactant liquor pegylation reaction rate equate or differ ± 1.5%.
Merge-checking of S4: according to the absorbance descent coefficient K of each reactant liquor
260comparable situation, each reactant liquor that can be admixed together merges and obtains amalgamation liquid, then uses the pegylation reaction rate of high performance liquid chromatograph combining data detection liquid.
The testing conditions that the above high performance liquid chromatography detects is: mobile phase is that concentration is 50mM, the phosphate buffer that pH is 6.8; Detection wavelength is 280nm; Flow velocity is 0.5mL/min.
Compared with prior art, the invention has the beneficial effects as follows: the present invention is developer by using nano gold sol, can improve significantly the ultraviolet absorptivity of reactant liquor, obviously improved the sensitivity detecting.And reactant liquor is carried out to multistage dilution, can reduce the impact that in reactant liquor, PEG detects ultraviolet, thereby improve the accuracy of testing result.Calculate absorbance descent coefficient, the PEGization reaction rate of more each reactant liquor fast according to testing result; Compare in conjunction with the testing result of the HPLC of one of them reactant liquor again, PEGization reaction rate that can the each reactant liquor of Rapid identification, thus can judge whether fast to merge corresponding reactant liquor.The HPLC that can reduce in PEG-EPO medicine production process by detection method of the present invention detects number of times, therefore can greatly shorten the detection analysis time before reactant liquor merges, improve the efficiency of PEG-EPO medicine production technology, saved human cost and time cost.And detection method of the present invention has good sensitivity, reliability and reappearance, can be applicable in the production of PEG-EPO medicine.
Brief description of the drawings
Fig. 1 is the PEGization reaction equation (using the PEG reagent with methyl end groups) of EPO;
Fig. 2 is the HPLC testing result of the PEGization reactant liquor of experiment one gained EPO;
The serve as reasons MS testing result of the isolated EPO of PEGization reactant liquor of experiment one EPO of Fig. 3;
The serve as reasons MS testing result of the isolated mPEG-EPO product of PEGization reactant liquor of experiment one EPO of Fig. 4;
Fig. 5 is the electrophoresis detection result of the PEGization reactant liquor of the EPO of experiment one;
Fig. 6 is the circular dichroism spectra testing result of the PEGization reactant liquor of the EPO of experiment one;
Fig. 7 is the UV-Vis testing result of the PEGization reactant liquor of the EPO of experiment one;
Fig. 8 is the UV-Vis testing result of NGP;
Fig. 9 is the TEM testing result (scale is 100nm) of NGP;
Figure 10 is the TEM testing result (scale is 20nm) of NGP;
Figure 11 is the TEM testing result of NGP in pH7.0PBS;
Figure 12 is the potpourri of PEGization reactant liquor of NGP and the EPO TEM testing result in pH7.0PBS;
Figure 13 is the PEGization reactant liquor of NGP and the EPO UV-Vis testing result in pH7.0PBS;
Figure 14 is the PEGization reactant liquor of NGP and the EPO CD testing result in pH7.0PBS;
Figure 15 is NGP and the CD testing result of EPO stoste in pH7.0PBS;
Figure 16 is NGP and the UV-Vis testing result of PEG in pH7.0PBS;
Figure 17 is the UV-Vis testing result that quantitatively detects PEG solution concentration taking NGP as developer;
Figure 18 is NGP and the UV-Vis testing result of EPO in pH7.0PBS;
Figure 19 is the UV-Vis testing result that quantitatively detects EPO solution concentration taking NGP as developer;
Figure 20 is NGP and the UV-Vis testing result of mono-PEG-EPO in pH7.0PBS;
Figure 21 is the UV-Vis testing result of the disturbing effect of additional EPO solution to PEG detection;
Figure 22 is the UV-Vis testing result of EPO and PEG disturbing effect each other;
Figure 23 is the UV-Vis testing result of the multistage dilution experiment of PEGization reactant liquor of EPO.
Embodiment
In order to more fully understand technology contents of the present invention, below in conjunction with specific embodiment, technical scheme of the present invention is described further and is illustrated.
The main chemical reagent that the present invention uses comprises: sodium dihydrogen phosphate (NaH
2pO
42H
2o, pharmaceutical grade); Sodium hydrogen phosphate (Na
2hPO
42H
2o, pharmaceutical grade); NaOH (NaOH, pharmaceutical grade); Hydrochloric acid (HCl analyzes pure); Gold chloride (HAuCl
4, be for No. CAS: 16903-35-8, molecular weight is: 339.79g/mol); Trisodium citrate (C
6h
5na
3o
72H
2o, analyzes pure).
EPO stoste is from Shenzhen Sciprogen Biology Medicine Co., Ltd; PEG reagent (GLUC-PEG-NHS, mean molecular weight is 36097Da), from Jiankai Science and Technology Co., Ltd., Beijing.
The PEGization reaction of experiment one: EPO
Use 50mL plastic centrifuge tube, add 4.89mLEPO stoste (protein content is 3.07mg/mL, altogether 15mg), and the 40mM phosphate buffer of 10.11mLpH7.0, the final concentration of EPO is 1mg/mL.Accurately weigh 300mg GLUC-PEG-NHS, add the phosphate buffer of 3.0mL2mM and pH3.0 to dissolve.After dissolving, GLUC-PEG-NHS solution is all added in EPO, mix rapidly.Gained mixed liquor is placed on oscillator, room temperature oscillating reactions 1 hour.Thus, obtain PEG-EPO reactant liquor colourless, transparent, clarification.For subsequent use.
Experiment two: the HPLC of the PEGization reactant liquor of EPO detects
Taking the PEGization reactant liquor of EPO of testing a gained as sample, sampling is made HPLC and is detected.Testing conditions is: GE superpose6 analytical column; Shimadzu HPLC, PDA detecting device, LC Solution workstation; PBS damping fluid is made mobile phase, and concentration is 50mM, and pH is 6.8; Detection wavelength is 280nm; Applied sample amount is 20 μ L; Flow velocity is 0.5mL/min.Testing result as shown in Figure 2.Can find from Fig. 2, in the PEG-EPO of gained reactant liquor, except free EPO (retention time is: 34.043min), also have at least three kinds of different PEG-EPO products (retention time is respectively: 25.318min (monosubstituted), 21.416min (two replace) and 14.876min (three replace)), and its molecular polarity is more approaching.Peak area with Fig. 2 carries out integral and calculating, and result shows, the free EPO content in the PEGization reactant liquor of gained EPO is about 25%, and monosubstituted rate is about 50%, and polysubstituted rate is about 25%.That is to say, in this reaction, the PEGization reaction rate of EPO is about 75%.
Experiment three: the Mass Spectrometer Method of free EPO and mPEG-EPO product
According to the testing result (Fig. 2) of experiment two, the PEGization reactant liquor of the EPO of experiment one gained is carried out to purification process, obtain pure free EPO (HPLC retention time is: 34.043min) and mPEG-EPO product (HPLC retention time is: 25.318min), then mass spectrum (MS) detection is done in sampling respectively.Examination criteria is two annex IX J mass spectroscopies of Chinese Pharmacopoeia (2010 editions), and detecting instrument is MALDI-TOF-MS.Testing result as shown in Figure 3 and Figure 4.Can find from Fig. 3, Fig. 4, the free EPO that purifying obtains and the Mass Spectrometer Method result of mPEG-EPO product respectively: 29673 and 65131.Meanwhile, consider that the mean molecular weight of the PEG reagent in experiment one is 36097, therefore proved that the PEGization reaction of EPO is successfully, and mPEG-EPO product is the primary product of reaction.
Experiment four: the electrophoresis detection of the PEGization reactant liquor of EPO
Taking the PEGization reactant liquor of EPO of testing a gained as sample, electrophoresis detection is made in sampling.Detect by Chinese Pharmacopoeia method, testing result as shown in Figure 5.The result of Fig. 5 shows, in the PEGization reactant liquor of EPO, except free EPO (molecular weight is 30KD～45KD), also have at least two kinds of different PEG-EPO products (molecular weight be 66KD～106KD (monosubstituted), be greater than 106KD (two replace)).Because PEG can expand in solution, thereby cause the molecular weight of PEG-EPO molecule on electrophoretogram can be larger than actual molecular weight, therefore the HPLC testing result of the electrophoresis result of Fig. 5 and Fig. 2 be basically identical.
Experiment five: the circular dichroism spectra of the PEGization reactant liquor of EPO detects
To test one EPO stoste, and the PEGization reactant liquor of the EPO of gained is sample, and sampling is made circular dichroism spectra (CD) and detected.Detect according to a conventional method, detecting instrument is Chirascan type circular dichroism spectrometer, and testing result as shown in Figure 6.Can find from Fig. 6, the circular dichroism spectra of PEG-EPO reactant liquor and EPO stoste is closely similar, and the secondary protein structure of two samples is very approaching.This PEGization that EPO has been described is reacted, and does not change significantly the secondary protein structure of EPO.
Experiment six: the uv-visible absorption spectroscopy of the PEGization reactant liquor of EPO detects
Taking the PEGization reactant liquor of EPO of testing a gained as sample, sampling is made uv-visible absorption spectroscopy (UV) and is detected.Detect according to Chinese Pharmacopoeia method, sweep limit is 200～700nm, and testing result as shown in Figure 7.Can find from Fig. 7, the PEGization reactant liquor of EPO does not have obvious absorption peak in visible region, has a significant absorption peak simultaneously at ultraviolet region 260 ± 1nm place.List of references (Yang Ruie etc., PEGYLATED G-CSF's technical research), this ultraviolet absorption peak is produced by PEG group, comprises the peg moiety in PEG molecule and the PEG-EPO product of not being combined with EPO.
Experiment seven: the PEGization reactant liquor of preparing and detect the EPO of different PEGization reaction rates
Detect the feasibility of the PEGization reaction rate of EPO in order to study application uv-visible absorption spectroscopy (UV-Vis), first prepared 5 kinds and detected the PEGization reactant liquor that proves the EPO with different PEGization reaction rates through HPLC, then UV-Vis detection is done in sampling.
The method of PEGization reactant liquor of EPO that preparation has different PEGization reaction rates is as follows: with to test one basically identical, difference is respectively to add in the 1mg/mLEPO solution before reaction 0.00,0.05,0.10,0.15 and the 40mM ammonium bicarbonate soln of 0.20mL, mixes.After completion of the reaction, HPLC test (by the method for experiment two) and UV-Vis test (by the method for experiment six) are done in sampling respectively, the results are shown in table 1.
The HPLC of the PEGization reactant liquor of the EPO of the different PEGization reaction rates of table 1 and UV-Vis testing result
Can find from the HPLC testing result of table 1, along with the increase of ammonium bicarbonate soln addition, having there is change in the PEGization reaction rate of EPO, gradually drops to 57.11% from 76.19% significantly, and the free EPO content in gained PEG-EPO reactant liquor has finally improved and approaches twice.Meanwhile, the monosubstituted rate of reaction and the ratio of polysubstituted rate also increase substantially, and have risen to 3.9:1.0 by 1.9:1.0, and final polysubstituted rate has been in close proximity to 10%.This has fully proved, can, by adding micro-ammonium sulfate, regulate the PEGization reaction result of EPO, especially in the monosubstituted rate that maintains reaction in higher level, significantly reduce the polysubstituted rate of reacting.And 5 kinds of different PEG-EPO reactant liquors of gained are well suited for doing to detect sample, to verify that application UV-Vis method detects the feasibility of the PEGization reaction rate of EPO.
But, the UV-Vis testing result of table 1 shows, it is infeasible directly applying the PEGization reaction rate that UV-Vis method detects EPO.Because while variation in about 20% scope along with the PEGization reaction rate of the EPO being determined by HPLC, although corresponding ultra-violet absorption spectrum has consistent maximum absorption band, but there is not obvious change in its corresponding absorbance, does not have the variation of rule.Its main cause should be because in the PEGization reactant liquor of gained EPO, has a large amount of PEG molecules of not being combined with EPO, and they have conclusive impact to the absorbance of ultraviolet spectrum.
Therefore,, if these the 5 kinds difference UV-Vis methods with the PEGization reactant liquor of the EPO of different PEGization reaction rates need to be detected, so just need to adopt some new detection skill and data processing methods.Comprise: 1) in the PEGization reactant liquor of EPO, add phosphate buffer, thereby form stable color development system; 2) the PEGization reactant liquor of EPO is carried out to dilution process, impact UV-Vis being detected to reduce unreacted PEG molecule, thereby the accuracy that improves UV-Vis detection method; 3) nm of gold (Nano-gold particles, NGP) of introducing the PEGization reactant liquor absorbance that can strengthen EPO is made developer, thereby improves the sensitivity of UV-Vis detection method.
Experiment eight: preparation and the detection thereof of nm of gold (NGP) colloidal sol
Adopt Frens method (Nat Phys Sci, 1973,241:20.), i.e. the trisodium citrate water reducing process of gold chloride, preparation NGP colloidal sol.Experimental technique is as follows: 1) a 250mL beaker is positioned on magnetic stirring apparatus, adds wherein 1.0mL1% gold chloride storing solution, and 99.0mL water for injection, fully stir, obtain 0.01% gold chloride reactant liquor; 2) start heating, gained gold chloride reactant liquor is heated to boiling; 3) the rapid disposable 2.0mL1.0% citric acid three sodium solution that adds; 4) by gained mixed liquor continuous heating 15min; 5) stop heating, allow it naturally cool to room temperature; 6) after treating that gained nm of gold is fully cooling, be transferred in plastic centrifuge tube, keep in Dark Place.For subsequent use.Gained NGP colloidal sol is peony, transparence.
Within the scope of 200-800nm, taking water for injection as reference, scan its uv-visible absorption spectroscopy, result is as shown in Figure 8.Meanwhile, adopt transmission electron microscope (TEM) to detect its microscopic pattern and particle size, result is as shown in Fig. 9 (scale is 100nm) and Figure 10 (scale is 20nm).Can find from Fig. 8, the maximum absorption band of gained NGP is positioned at 520nm place; , can find from the TEM result of Fig. 9 and Figure 10, its particle diameter is about 20nm meanwhile, and micromechanism is good.
In experiment 9-16 below and embodiment, NGP colloidal sol used is to be all 3.0 × 10 by the prepared concentration of method of experiment 8
-9the NGP colloidal sol of mol/L.
Experiment nine: the interaction of the PEGization reactant liquor of NGP and EPO
Adopt transmission electron microscope (TEM), ultraviolet-visible spectrum (UV-Vis) and circular dichroism spectra (CD) technology, study at phosphate buffer (pH7.0PBS, concentration is 40mM) in environment, interaction between the PEGization reactant liquor of NGP and EPO, result is respectively as shown in Figure 11, Figure 12, Figure 13, Figure 14 and Figure 15.
Can find from the TEM testing result of Figure 11 and Figure 12, NGP is obvious disperse state in pH7.0PBS, and when toward after wherein adding the PEGization reactant liquor of EPO, NGP presents state of aggregation more closely.This has illustrated between the PEGization reactant liquor of NGP and EPO and has existed and interact, and in the PEGization reactant liquor of EPO, composition can make the NGP in disperse state again assemble agglomerating effectively.This process also shows as the variation of NGP solution colour, and NGP colloidal sol is peony originally, presents pale red in pH7.0PBS solution, even can present light blue after being long placed in.And after it is mixed mutually with the PEGization reactant liquor of EPO, gained mixed solution is red.This is mainly because produced electrostatic attraction effect.
The UV-Vis testing result of Figure 13 shows, after NGP mixes mutually with the PEGization reactant liquor of EPO, increases substantially in the absorbance of the maximum absorption band at 520nm place.Meanwhile, the PEGization of EPO reactant liquor has also promoted and has exceeded 50% in the absorbance of the maximum absorption band at 260nm place.This has fully confirmed, has significant interaction between NGP and PEG-EPO reactant liquor, and NGP can improve the ultraviolet absorptivity of PEG-EPO reactant liquor significantly.
The CD testing result of Figure 14 and Figure 15 has shown, the interaction between the PEGization reactant liquor of NGP and EPO is relevant with epo protein.Can find from Figure 14, after adding NGP, the unique negative peak on the CD spectrum of the PEGization reactant liquor of EPO by 214nm red shift to 219nm; , can find from Figure 15 meanwhile, after adding NGP, the unique negative peak on the CD spectrum of EPO stoste by 212nm red shift to 215nm.This is mainly to have produced comparatively faint bonding action because of groups such as amino, sulfydryls on NGP and epo protein matter molecule.In addition, the result of Figure 14 and Figure 15 also illustrated, NGP can not destroy protein component in the PEGization reactant liquor of EPO tempestuously, comprises the secondary protein structure of target product (the monosubstituted product of PEG-EPO).This has ensured to adopt NGP to make reliability and the reappearance of the analytical approach of developer detection PEG-EPO reactant liquor.
Experiment ten: detect PEG taking NGP as developer
In 40mMpH7.0PBS buffer environment, detect PEG taking NGP as developer.Experimental technique is: get two 10mL color comparison tubes, add first respectively 4.0mLPBS solution, then add the PEG solution of 2.0mL16mg/mL toward 1 color comparison tube wherein; Add PEG solution and the 2.0mLNGP colloidal sol of 2.0mL16mg/mL toward other 1 color comparison tube.Finally, be diluted to scale with water for injection, ie in solution cumulative volume is 10mL.Fully mix, after colour developing 10min, taking 40mMpH7.0PBS as blank, within the scope of 220-700nm, the uv-visible absorption spectroscopy of scanning gained sample, result is as shown in figure 16.The PEG solution that is 16mg/mL concentration, making concentration range into is 20 kinds of PEG solution of 0.1mg/mL～20mg/mL, repeats above test, determines the range of linearity that quantitatively detects PEG solution concentration taking NGP as developer, testing result is as shown in table 2 and Figure 17.
The UV-Vis testing result of Figure 16 shows, NGP can improve the absorbance of PEG at 260nm place absorption peak significantly, thereby increased the sensitivity that detects PEG with UV-Vis method.Simultaneously, the testing result of the variable concentrations PEG solution of Figure 17 shows, in the concentration range of 1mg/mL～20mg/mL, the concentration of PEG solution becomes good linear relationship with the absorbance at 260nm place, further calculates corresponding linear equation and related coefficient and is: A
260=0.0511c
pEG+ 0.2387, R
2=0.9979.And along with the variation of PEG solution concentration, NGP does not have obvious linear changing relation in the absorbance at 520nm place, is not suitable for use in quantitative detection.Due in the production run of PEG-EPO medicine, about the PEG concentration range of solution is generally 1mg/mL～18mg/mL, therefore can apply income approach to its PEG concentration, carry out the quantitative detection directly or through diluting.
The UV-Vis testing result of the PEG solution of table 2 variable concentrations
Test 11: detect EPO taking NGP as developer
In 40mMpH7.0PBS buffer environment, detect EPO taking NGP as developer.Experimental technique is: get two 10mL color comparison tubes, add first respectively 4.0mLPBS solution, then add the EPO solution of 2.0mL0.8mg/mL toward 1 color comparison tube wherein; Add EPO solution and the 2.0mLNGP colloidal sol of 2.0mL0.8mg/mL toward other 1 color comparison tube.Finally, be diluted to scale with water for injection, ie in solution cumulative volume is 10mL.Fully mix, after colour developing 10min, taking 40mMpH7.0PBS as blank, within the scope of 220-700nm, the uv-visible absorption spectroscopy of scanning gained sample, result is as shown in figure 18.The EPO solution that is 0.8mg/mL concentration, making concentration range into is 20 kinds of EPO solution of 0.01mg/mL～2mg/mL, repeats above test, determines the range of linearity that quantitatively detects EPO solution concentration taking NGP as developer, testing result is as shown in table 3 and Figure 19.
The UV-Vis testing result of Figure 18 shows, NGP can improve the absorbance of the absorption spectrum of EPO significantly, thereby increased the sensitivity that detects EPO with UV-Vis method.But, the original maximum absorption band that is positioned at 260nm place of EPO has produced significant blue-shifted phenomenon after mixing with NGP, shows to have produced between EPO and NGP significant interaction.Meanwhile, the testing result of the variable concentrations EPO solution of Figure 19 shows, along with the variation of EPO solution concentration, the variation of obvious linear relationship has occurred to have in the absorbance at 520nm place NGP, can be as quantitatively detecting.Further research is found, in the concentration range of 0.05mg/mL～0.15mg/mL, the concentration of EPO solution becomes good linear relationship with the absorbance at 520nm place, and corresponding linear equation and related coefficient are: A
520=0.4c
ePO+ 0.3156, R
2=0.9902.Due in the production run of PEG-EPO medicine, about the EPO concentration range of solution is generally 0.2mg/mL～2.0mg/mL, therefore can apply income approach to its EPO concentration, carry out the quantitative detection after dilution.
The UV-Vis testing result of the PEG solution of table 3 variable concentrations
| Concentration (mg/mL) | 0.05 | 0.08 | 0.10 | 0.15 |
| Absorbance | 0.333 | 0.348 | 0.355 | 0.365 |
Test 12: detect PEG-EPO product taking NGP as developer
In 40mM pH7.0PBS buffer environment, detect the target product of the PEGization reaction of EPO taking NGP as developer, i.e. the monosubstituted thing of PEG-EPO (mono-PEG-EPO).Experimental technique is: get two 10mL color comparison tubes, add first respectively 4.0mL PBS solution, then add the mono-PEG-EPO solution obtaining of 2.0mL0.25mg/mL toward 1 color comparison tube wherein after purifying process is processed; Add mono-PEG-EPO solution and the 2.0mL NGP colloidal sol of 2.0mL0.25mg/mL toward other 1 color comparison tube.Finally, be diluted to scale with water for injection, ie in solution cumulative volume is 10mL.Fully mix, after colour developing 10min, taking 40mMpH7.0PBS as blank, within the scope of 220-700nm, the uv-visible absorption spectroscopy of scanning gained sample, result is as shown in figure 20.
The UV-Vis testing result of Figure 20 shows, NGP can improve the absorbance of the absorption spectrum of mono-PEG-EPO product significantly, thereby increased the sensitivity that detects mono-PEG-EPO with UV-Vis method.Relatively Figure 18 and Figure 20 can find, mono-PEG-EPO product and EPO stoste, have quite similar absorption spectrum feature, do not occur the characteristic absorption peak of PEG at 260nm place.This has illustrated that the chromophore on PEG molecule has been subject to sheltering after PEG and EPO carry out combination with chemical bond form.Therefore, can apply the absorption spectroscopy for EPO, mono-PEG-EPO is carried out effectively quantitatively detecting.Meanwhile, this has also illustrated, in the time that application NGP system is carried out UV-Vis detection to PEG-EPO reactant liquor, except unreacted epo protein, reacts the PEG-EPO substituent generating and also can produce similar absorbance variable effect to gained spectrogram; And the absorptivity of related protein composition is discrepant, be difficult to this to carry out quantitative test.So, the result of Comprehensive Experiment ten, experiment 11 and experiment 12, in the time that application NGP system detects analysis to PEG-EPO reactant liquor, the absorbance variation that is positioned at the 260nm place of ultraviolet region is comparatively valuable testing result.
Test 13: the disturbing effect that EPO quantitatively detects PEG
In 40mMpH7.0PBS buffer environment, investigate while detecting PEG taking NGP as developer the disturbing effect of additional EPO.Experimental technique is: get 10 10mL color comparison tubes, first all add the PEG solution of 4.0mL PBS solution and 2.0mL12mg/mL, then adding respectively 2.0mL concentration toward 9 color comparison tubes is 0.2,0.5,0.8,1.0,1.5,2.0,2.5,3.0 and the EPO stoste of 3.5mg/mL, the remaining not additional EPO stoste of 1 color comparison tube.Finally, be diluted to scale with water for injection, ie in solution cumulative volume is 10mL.Fully mix, after colour developing 10min, taking 40mMpH7.0PBS as blank, within the scope of 220-700nm, scanning uv-visible absorption spectroscopy, result is as shown in figure 21.
Can find from Figure 21, along with the increase of additional EPO solution concentration, the absorbance of the characteristic absorption peak of PEG (260nm) also rises gradually, has finally reached 1.156 by original 0.798.This has illustrated, the quantitative detection existence comparatively serious disturbing effect of EPO to PEG.Therefore,, when using NGP as developer, when the PEG concentration in PEG-EPO reactant liquor is quantitatively detected, must consider the disturbing effect of EPO and PEG-EPO substitution product, otherwise the testing result obtaining is by unreliable.Meanwhile, clearly, the PEG in the PEGization reactant liquor of EPO also will produce very important disturbing effect to the quantitative detection of EPO concentration.
To this, there is further experimental result to verify, as shown in figure 22.Can find from Figure 22,0.83mg/mLEPO solution is mixed mutually with 16mg/mLPEG solution, gained mixed solution all obviously improves in the absorbance of 520nm and two absorption peaks of 260nm, thereby makes its absorbance at 520nm place equate with the simple corresponding absorbance of 1.0mg/mLEPO solution; Its absorbance at 260nm place equates with the absorbance of simple 20mg/mLPEG solution.Because the PEGization of the EPO reactant liquor mixed liquor that namely EPO solution and PEG solution mix in fact, so the result of Figure 22 has also shown, if EPO or PEG are quantitatively detected at the absorbance numerical value at 520nm or 260nm place according to the PEGization reactant liquor of EPO, will there is about 20% error in acquired results so.In addition, because the condition that the PEGization of actual EPO is reacted is different, cause the PEGization reaction rate of EPO inconsistent, the composition of PEGization reactant liquor that is gained EPO is non-constant, therefore directly EPO or PEG in the PEGization reactant liquor of EPO are quantitatively detected, there is larger error, and then cannot determine the PEGization reaction rate of EPO.
But the result of comprehensive Figure 21 and Figure 22, can find, the protein component in the PEGization reactant liquor of EPO, i.e. EPO, mono-PEG-EPO and mutil-PEG-EPO, all to PEG in the absorbance at the 260nm place effect that is significantly improved.In the situation that epo protein matter total amount is constant, the composition of the PEGization reactant liquor of the size of this raising effect and EPO, the PEGization reaction rate that is EPO has direct relation, and the PEGization of EPO reactant liquor is in the absorbance variation tendency at 260nm place, the composition of its epo protein matter can be indirectly reflected, thereby the PEGization reaction rate of corresponding EPO can be weighed.
Test 14: the absorbance of the PEGization reactant liquor of EPO improves coefficient test
First, prepare four kinds of PEGization reactant liquors with the EPO of different PEGization reaction rates, preparation method is one basically identical with experiment, and difference is respectively in 1mg/mL EPO solution, to add 0.05,0.10,0.15 and the 40mM glycine solution of 0.20mL toward reacting, and mixes.After completion of the reaction, HPLC test is done in sampling respectively, the results are shown in table 4.
The HPLC testing result of the PEGization reactant liquor of the EPO of the different PEGization reaction rates of table 4
PEGization reactant liquor to four kinds of EPO of gained has carried out UV-Vis detection, and all methods are similar to the method for experiment 12, and difference is as follows: get 8 10mL color comparison tubes, every 2 is one group, and totally 4 groups, corresponding to the PEGization reactant liquor of four kinds of different EPO; Wherein 1 PEGization reactant liquor that only adds EPO of every group of color comparison tube, does not add NGP, and other 1 PEGization reactant liquor and NGP that simultaneously adds EPO, detects the absorbance of gained color development system at 260nm place.Thus, study the absorbance variation of adding NGP front and back, and the ratio that corresponding absorbance is improved is decided to be absorbance raising coefficient, the as a comparison index of the PEGization of different EPO reactant liquor.Experimental result is listed in table 5.
Can find from the result of table 3, there is the PEGization reactant liquor of the EPO of different PEGization reaction rates (34.97%～79.21%), add NGP front and back all very approaching in the absorbance at 260nm place, gained absorbance improves coefficient and is also more or less the same, and is difficult to distinguish.This has illustrated, the PEGization reactant liquor of EPO is carried out to direct staticize detection, is difficult to obtain the feature of its PEGization reaction rate.Therefore, in order to apply easy UV-Vis method, the PEGization reactant liquor of EPO is effectively detected, need to carry out some technical finesses, by detecting the variation of 260nm place absorbance numerical value, the PEGization reaction rate of the PEGization reactant liquor of indirect detection EPO.
The absorbance of the PEGization reactant liquor of the EPO of the different PEGization reaction rates of table 5 improves coefficient experimental result
Test 15: the multistage dilution experiment of the PEGization reactant liquor of EPO
Due in the time that the PEGization of carrying out EPO is reacted, in order to maximally utilise comparatively expensive epo protein matter, conventionally all can add greatly excessive PEG reagent, the PEG reagent that contains abundant residues in the PEGization reactant liquor of gained EPO, therefore in the time that the PEGization reactant liquor to EPO carries out UV-Vis detection, PEG is main colour developing group, also can produce masking effect to the colour developing group of the protein component in reactant liquor (EPO, mono-PEG-EPO and mutil-PEG-EPO) simultaneously.So, for the PEGization reactant liquor to EPO effectively and accurately carries out UV-Vis detection, need to reduce the PEG concentration in detection system, and then allow the absorption spectrum feature of protein component be presented, therefore the PEGization reactant liquor of EPO being carried out to multistage dilution is a kind of effective method.
To test one method, again prepare the PEGization reactant liquor of the EPO of a collection of 15mgEPO reacting dose, then taking it as sample, carry out multistage dilution experiment, with water for injection, sample is diluted respectively after 2,4,6,8,10 times, refer again to the method for experiment ten, carry out UV-Vis detection, acquired results as shown in figure 23.
Can find from Figure 23, after the PEGization reactant liquor of EPO is carried out to multistage dilution, the absorbance at gained 520nm place does not significantly change, and presents the significant trend reducing step by step in the absorbance at 260nm place.This has illustrated in the time of NGP content constant in detection system, protein component in the PEGization reactant liquor of EPO is after 10 times of dilutions, still have enough content and NGP to maintain stable colour developing bond, remaining PEG composition does not also form stable colour developing bond with NGP.Further analyze and show, the absorbance of the 260nm after the PEGization reactant liquor stepwise dilution of EPO changes, and has good linear relationship with extension rate, and the protein component content size in simultaneous reactions liquid exists directly impact to this linear relationship.That is to say, in the time that the PEGization reactant liquor to EPO carries out stepwise dilution, have downtrending corresponding to the absorbance of the ultraviolet absorption peak of PEG, and protein component in reactant liquor has and significantly slows down effect to it.
To this, taking the PEGization reactant liquor of testing the EPO with different PEGization reaction rates in seven as sample, carry out experimental verification by above-mentioned multistage dilution process.Then, with the absorbance numerical value A at gained 260nm place
260carry out linear fit with extension rate, obtaining corresponding linear fit equation (is A
260=ax+b, x is extension rate).Meanwhile, by a value in gained linear fit equation (being slope of a curve), being multiplied by the numerical value obtaining that takes absolute value again after 10000 and being decided to be absorbance descent coefficient value (k
260), to make comparisons.Related results is listed in table 6 and table 7.
The UV-Vis testing result of the PEGization reactant liquor of the EPO of the different PEGization reaction rates of table 6
The linear fit equation of the PEGization reactant liquor of the different EPO of table 7 and absorbance descent coefficient
As can be found from Table 7, in the time that PEGization reaction rate is different, the PEGization reactant liquor of gained EPO approaches in the absorbance at 260nm place very much, namely cannot detect the difference of its protein component content, can only reflect the absorption spectrum feature of abundant residues PEG.And the absorbance descent coefficient (k that adopts multistage dilution method to obtain
260), very clearly demonstrate the characteristic ultraviolet absorption of the PEGization reactant liquor of the EPO of different PEGization reaction rates.In the time that the PEGization reaction rate of EPO is different, corresponding absorbance descent coefficient can produce significant variation.The variation of this absorbance descent coefficient, closely related with the protein component content in the PEGization reactant liquor of EPO, namely reflect EPO and the PEG-EPO product size that slows down effect to the downtrending of PEG absorbance.So the absorbance descent coefficient of the PEGization reactant liquor to gained EPO compares, and just can determine the difference of its protein component content, i.e. the difference of the PEGization of EPO reaction rate.See simply, the PEGization reaction rate of EPO is larger, and corresponding absorbance descent coefficient is less.This has illustrated, the free EPO content in the PEGization reactant liquor of EPO is larger, and the downtrending of the absorbance of PEG characteristic absorption peak is less.
Thus, set up the detection method of the PEGization reaction rate of a kind of new EPO.Income approach uses that instrument is simple, running program is easy, consuming time short, reliability is higher, can be applied to the mode large-scale production PEG-EPO medicine of employings " in a small amount repeatedly reaction " time, each reactant liquor is merged to the front fast detecting to reactant liquor.
Experiment 16: the PEGization reactant liquor that detects the EPO of different PEGization reaction rates with multistage dilution method
The preparation method of the PEGization reactant liquor of EPO, one basically identical with experiment, difference is respectively in 1mg/mL EPO solution, to add 0.00,0.05,0.10,0.15 and the 40mM ammonium sulfate of 0.20mL toward reacting, and mixes.After completion of the reaction, HPLC test (by the method for experiment two) is done in sampling respectively, the results are shown in table 8.Can find from the result of table 8, along with the increase of ammonium sulfate addition, the PEGization reaction rate of EPO significantly declines, and in the PEGization reactant liquor of gained EPO, free EPO content has finally improved and exceedes twice.Meanwhile, the monosubstituted rate of reaction also becomes multiple to improve with the ratio of polysubstituted rate, has finally brought up to 7.3:1.0, and polysubstituted rate is at this moment far below 10%.This has fully proved, can, by adding micro-ammonium sulfate, regulate the PEGization reaction result of EPO, especially in the monosubstituted rate that maintains reaction in higher level, significantly reduce the polysubstituted rate of reacting.
The HPLC testing result of the impact of the PEGization reaction of the micro-ammonium sulfate of table 8 on EPO
The multistage dilution method of application based on nm of gold, the PEGization reactant liquor 5 kinds of gained to the EPO of different PEGization reaction rates carries out UV-Vis detection.Selected 3 extension rates (x), respectively: 4,8,10.Adopt 10mL color comparison tube, buffering liquid used is 4.0mL40mMpH7.0PBS, and NGP addition is 2.0mL, and the addition unification of the PEGization reactant liquor of the EPO of process dilution is 2.0mL, is diluted to scale with water for injection.Fully mix, after colour developing 10min, taking 40mM pH7.0PBS solution as blank, detect the absorbance (A of sample at 260nm place
260), then according to experiment, the method described in 15 is made linear fit equation and is calculated the absorbance descent coefficient (k of each sample system
260).Experimental result is listed in table 9.The experimental result of table 9 shows, along with the PEGization reaction rate of the PEGization reactant liquor of EPO declines gradually, corresponding absorbance descent coefficient rises gradually, has clearly reflected the feature of the PEGization reactant liquor of each EPO.That is to say, gained UV-Vis testing result can be verified mutually with corresponding HPLC testing result.This has illustrated, the difference of the PEGization reactant liquor that the multistage dilution method based on NGP can detect different EPO effectively aspect PEGization reaction rate.In addition, the result of comprehensive comparison sheet 7 and table 9, can find, gained detection method has good accuracy and reappearance, is not subject to the disturbing effect of micro-additional substance in the PEGization reactant liquor of EPO.
The testing result of the multistage dilution experiment of the PEGization reactant liquor of the EPO of the different PEGization reaction rates of table 9
Embodiment
The multistage dilution UV-Vis detection method of testing above 15-16 is applied in the production run of PEG-EPO, before multiple reactant liquors merge, each reactant liquor is identified fast.
PEG according to the EPO of experiment one is combined to method, carry out continuously the PEGization reaction of 6 EPO, the reacting dose of EPO is respectively: 5mg (1 reaction), 200mg (1 reaction) and 400mg (4 reactions), the sequence number of the PEGization reactant liquor of gained EPO is decided to be respectively: 15,16,17,18,19,20.Except the pre-reaction that reacting dose is 5mg, the PEGization reactant liquor of all the other 5 EPO is through detecting the PEGization reactant liquor that needs to merge the EPO that is formed together a collection of 1800mg amount after qualified.Detection method before reactant liquor merges is as follows:
(1) pre-reaction that is 5mg to reacting dose sampling is made HPLC and is detected (identical with the method for experiment two).Testing conditions is: GE superpose6 analytical column; Shimadzu HPLC, PDA detecting device, LC Solution workstation; PBS damping fluid is made mobile phase, and concentration is 50mM, and pH is 6.8; Detection wavelength is 280nm; Applied sample amount is 20 μ L; Flow velocity is 0.5mL/min.Testing result is: monosubstituted rate 50.20%, polysubstituted rate 21.10%, free EPO content 28.70%, PEGization reaction rate 71.30%.
(2) the PEGization reactant liquor to 6 EPO and amalgamation liquid sampling thereof respectively, carries out UV-Vis detection.Selected 3 extension rates, respectively: 4,8,10.Adopt 10mL color comparison tube, buffering liquid used is 4.0mL 40mMpH7.0PBS, and concentration is 3.0 × 10
-9the NGP addition of mol/L is 2.0mL, and the addition unification of the PEGization reactant liquor of the EPO of process dilution is 2.0mL, is diluted to scale with water for injection.Fully mix, after colour developing 10min, taking 40mM pH7.0PBS solution as blank, detect the absorbance (A of sample at 260nm place
260), then carry out linear fit with absorbance numerical value and the extension rate at gained 260nm place, obtain corresponding linear fit equation (A
260=ax+b, the extension rate that x is reactant liquor).Meanwhile, the rate of curve in gained linear fit equation is multiplied by the numerical value obtaining that takes absolute value again after 10000 and is decided to be absorbance descent coefficient value (k
260).Gained testing result is listed in table 10.
(3) k of the reactant liquor that is 16-20 by sequence number
260value and sequence number are the k of 15 reactant liquor
260value compares, because of the k of each reactant liquor
260be worth identical or close, so each reactant liquor can be merged together.If (the k of certain reactant liquor
260value differs more and should not directly this reactant liquor and other reactant liquor be merged.)
(4) sampling of the reactant liquor after being combined by the method for step (1) is made HPLC and is detected, and testing result is: monosubstituted rate 50.30%, polysubstituted rate 21.15%, free EPO content 28.55%, PEGization reaction rate 71.45%.
The testing result of the application of the multistage dilution method of table 10 in the production run of PEG-EPO
The time of detecting due to each HPLC is approximately 1h, and adopt above detection method only need carry out HPLC detection by the reactant liquor to the reactant liquor of pre-reaction and after merging, and replace the HPLC of partial reaction liquid to detect by the detection of UV-Vis (multistage dilution method), thereby reduce the number of times that HPLC detects, therefore greatly shortened the detection analysis time of reactant liquor before merging, detect and become and be less than 3h from approaching 7h analysis time, improve well the efficiency of PEG-EPO medicine production technology, saved many human costs and time cost.
More than testing NGP colloidal sol used in 9-16 and embodiment can be to be also 1.0 × 10 by the prepared concentration of method of experiment 8
-11-6.0 × 10
-9the NGP colloidal sol of mol/L.
Comparative example
Carry out continuously the PEGization reaction of 6 EPO by the method for above-described embodiment, the reacting dose of EPO is respectively: 5mg (1 reaction), 200mg (1 reaction) and 400mg (4 reactions), the sequence number of the PEGization reactant liquor of gained EPO is decided to be respectively: 21,22,23,24,25,26.
In the production run of the PEG-EPO of prior art, before merging, differential responses liquid conventionally adopt HPLC respectively each reactant liquor to be detected.Detection method before reactant liquor merges is as follows:
(1) 6 reactant liquors are sampled respectively and do HPLC detection, testing conditions is identical with the HPLC testing conditions in embodiment.Testing result is listed in table 11.
(2) the HPLC testing result of more each reactant liquor, because the testing result of each reactant liquor is identical or close, so each reactant liquor can be merged together.If (testing result of certain reactant liquor differs more and should not directly this reactant liquor and other reactant liquor be merged.)
(3) sampling of the reactant liquor after being combined by the method for step (1) is made HPLC and is detected, and testing result is listed in table 11.
The testing result of the application of table 11HPLC in the production run of PEG-EPO
The time of detecting due to each HPLC is approximately 1h, and before merging, must detect the PEGization reactant liquor of 6 EPO of gained, therefore the HPLC of the production process of whole PEG-EPO has exceeded 7h detection time, thereby produce very important time cost and human cost, increased the production cost of PEG-EPO medicine.
In addition, shown by the experimental result of experiment nine, the interaction between NGP and EPO is the bonding action based between the groups such as amino, sulfydryl on epo protein matter molecule and NGP.Specifically, both can there is non-covalent Electrostatic Absorption with amino in NGP surface, can form very strong covalent bond with sulfydryl again.And most of protein molecule is all rich in amino group, and often with mercapto groups, therefore can predict NGP and also can form similar color development system to other oroteins molecule, and then detection method of the present invention can be applied to the spectroscopic methodology detection of the PEGization reactant liquor of most of other oroteins.This is consistent with bibliographical information, as: (1) Chen Xianguang etc., based on the Hydrogen Peroxide Biosensor of nm of gold and thionine immobilized enzyme, chemical journal, 2007,65 (4): 337-343; (2) Liu Xue equality, based on the fixing haptenic electrochemical immunosensor Sensitive Detection clenbuterol of nm of gold, analytical chemistry, 2012,40 (8): 1147-1152; (3) Sun Liping etc., the interaction of golden nanometer particle and single stranded DNA, SCI, 2009,30 (1): 95-99.
The above only further illustrates technology contents of the present invention with embodiment, so that reader is easier to understand, but does not represent that embodiments of the present invention only limit to this, and any technology of doing according to the present invention is extended or recreation, is all subject to protection of the present invention.
Claims (10)
1. a spectrum detection method for Recombinant Human Erythropoietin pegylation reaction liquid, is characterized in that, comprises the following steps:
S1 dilution: get n part reactant liquor sample, water dilutes reactant liquor sample by different extension rates, obtain the dilution of n kind variable concentrations, n≤3;
S2 configuration detection system: each dilution is mixed in different color comparison tubes from phosphate buffer and nano gold sol, and water is settled to scale and obtains having the detection liquid of variable concentrations, more than then detecting liquid colour developing 5min; The concentration of described phosphate buffer is 10-60mM, and pH is 5.0-8.0; The concentration of described nano gold sol is 1.0 × 10
-11-6.0 × 10
-9mol/L; The volume ratio of described phosphate buffer, nano gold sol and dilute reaction solution is 2:1:1;
S3, detection: survey the absorbance A of each detection liquid at 260nm place with ultraviolet spectrometer (UVS)
260;
S4, data processing: by the absorbance A of each detection liquid
260and extension rate carries out linear fit, obtain matched curve A
260=ax+b, x is extension rate; That gets a thoroughly deserves absorbance descent coefficient k
260.
2. the spectrum detection method of Recombinant Human Erythropoietin pegylation reaction liquid according to claim 1, is characterized in that, described in step S1 and step S2, water is water for injection.
3. the spectrum detection method of Recombinant Human Erythropoietin pegylation reaction liquid according to claim 2, is characterized in that, extension rate described in step S1 is respectively 4 times, 8 times and 10 times.
4. the spectrum detection method of Recombinant Human Erythropoietin pegylation reaction liquid according to claim 3, is characterized in that, the concentration of phosphate buffer described in step S2 is 40mM, and pH is 7.0.
5. the spectrum detection method of Recombinant Human Erythropoietin pegylation reaction liquid according to claim 4, is characterized in that, the concentration of nano gold sol described in step S2 is 3.0 × 10
-9mol/L.
6. the spectrum detection method of Recombinant Human Erythropoietin pegylation reaction liquid according to claim 5, is characterized in that, described in rapid S2, the particle diameter of nano gold sol is 15-25nm.
7. the spectrum detection method of Recombinant Human Erythropoietin pegylation reaction liquid according to claim 6, is characterized in that, detects liquid colour developing 5-20min described in step S2.
8. the rapid identification method before multiple Recombinant Human Erythropoietin pegylation reaction liquid merging, is characterized in that, comprises the following steps:
S1 ultraviolet detects: each reactant liquor is carried out respectively to dilution as claimed in claim 1, configuration detection system, detection and four steps of data processing successively, obtain the absorbance descent coefficient k of each reactant liquor
260;
S2 high performance liquid chromatography detects: the pegylation reaction rate that detects one of them reactant liquor with high performance liquid chromatograph; This reactant liquor is benchmark reactant liquor;
S3, comparison: by the absorbance descent coefficient k of other reactant liquor
260absorbance descent coefficient k with benchmark reactant liquor
260relatively, there is identical absorbance descent coefficient k
260reactant liquor pegylation reaction rate equate or differ ± 1.5%.
9. the rapid identification method of a kind of multiple Recombinant Human Erythropoietin pegylation reaction liquid before merging according to claim 8, it is characterized in that, also comprise merging-verification step, merge and obtain amalgamation liquid by different reactant liquors, then use the pegylation reaction rate of high performance liquid chromatograph combining data detection liquid.
10. the rapid identification method of a kind of multiple Recombinant Human Erythropoietin pegylation reaction liquid before merging according to claim 9, it is characterized in that, the testing conditions of described high performance liquid chromatography is: mobile phase is that concentration is 50mM, the phosphate buffer that pH is 6.8; Detection wavelength is 280nm; Flow velocity is 0.5mL/min.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0921817B1 (en) * | 1997-01-29 | 2001-03-28 | PolyMASC Pharmaceuticals plc | Pegylation process |
| EP1834963A1 (en) * | 2006-03-13 | 2007-09-19 | Siegfried Ltd. | Di-polymer protein conjugates and processes for their preparation |
| CN101614659A (en) * | 2009-07-24 | 2009-12-30 | 上海现代中医药技术发展有限公司 | The Chinese medicine leaching process is carried out the apparatus and method of online detection and control |
| CN102156174A (en) * | 2011-05-06 | 2011-08-17 | 吉林大学 | Detection of phosphatidylcholine in yolk lecithin by using high performance liquid chromatography |
| CN102854160A (en) * | 2012-08-31 | 2013-01-02 | 贵州泰邦生物制品有限公司 | Method for detection of biological activity of Fc region of human immune globulin through ultraviolet spectrophotometry |
| US20140011964A1 (en) * | 2007-02-28 | 2014-01-09 | Serina Therapeutics, Inc. | Activated Polyoxazolines and Conjugates and Compositions Comprising the Same |
-
2014
- 2014-04-18 CN CN201410159182.7A patent/CN104007079B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0921817B1 (en) * | 1997-01-29 | 2001-03-28 | PolyMASC Pharmaceuticals plc | Pegylation process |
| EP1834963A1 (en) * | 2006-03-13 | 2007-09-19 | Siegfried Ltd. | Di-polymer protein conjugates and processes for their preparation |
| US20140011964A1 (en) * | 2007-02-28 | 2014-01-09 | Serina Therapeutics, Inc. | Activated Polyoxazolines and Conjugates and Compositions Comprising the Same |
| CN101614659A (en) * | 2009-07-24 | 2009-12-30 | 上海现代中医药技术发展有限公司 | The Chinese medicine leaching process is carried out the apparatus and method of online detection and control |
| CN102156174A (en) * | 2011-05-06 | 2011-08-17 | 吉林大学 | Detection of phosphatidylcholine in yolk lecithin by using high performance liquid chromatography |
| CN102854160A (en) * | 2012-08-31 | 2013-01-02 | 贵州泰邦生物制品有限公司 | Method for detection of biological activity of Fc region of human immune globulin through ultraviolet spectrophotometry |
Non-Patent Citations (5)
| Title |
|---|
| GIANFRANCO PASUT, FRANCESCO M. VERONESE: "State of the art in PEGylation: The great versatility achieved after forty years of research", 《JOURNAL OF CONTROLLED RELEASE》 * |
| JUERGEN H. NETT ET AL.: "Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris", 《JOURNAL OF BIOTECHNOLOGY》 * |
| KATJA REBOLJ ET AL.: "Characterization of a Protein Conjugate Using an Asymmetrical-Flow Field-Flow Fractionation and a Size-Exclusion Chromatography with Multi-Detection System", 《ANALYTICAL CHEMISTRY》 * |
| 周勇等: "PEG 化重组人促红素质量控制方法和标准研究", 《药物分析杂志》 * |
| 张雪艳等: "PEG-EPO中残余聚乙二醇的碘染色法", 《药物分析杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112986368A (en) * | 2021-02-18 | 2021-06-18 | 华润昂德生物药业有限公司 | Method for detecting electric charge heterogeneity of polyethylene glycol recombinant human erythropoietin |
| CN112986368B (en) * | 2021-02-18 | 2023-08-22 | 华润昂德生物药业有限公司 | Detection method for charge heterogeneity of PEGylated recombinant human erythropoietin |
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